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Membrane proteins provide critical roles in cell-to-cell contact, surface recognition, cytoskeleton contact, signaling, enzymatic activity, or transporting substances across the membrane. However, membrane proteins are challenging because their expression can be toxic to cells or cause inclusion bodies, which limit protein yield, and current methods require tedious optimization making purification difficult and time consuming. We report a cell-free approach for expressing and inserting integral membrane proteins into planar phospholipid membrane bilayer surrounded by a scaffold protein (also called a nanolipoprotein particle or NLP; brand name MembraneMax). Proteins are inserted into these particles, circumventing the need of extracting and reconstituting the product into membrane vesicles. Moreover, the planar nature of the membrane support makes the protein freely accessible from both sides of the lipid bilayer.
Materials and MethodsAs described in J. Proteome Res. http://pubs.acs.org/cgi-bin/abstract.cgi/jprobs/asap/abs/pr800265f.html.
Results and DiscussionWe studied the in vitro expression and solubility of a host of membrane proteins of different topologies, sizes, origins, and proposed roles. Sixty-four percent of the proteins analyzed expressed at a level >0.1 mg/mL of the reaction, and, without exception, the proteins significantly increased their solubility in the presence of the MembraneMax reagent (Table 1 and Figure 1). Our method for the expression of integral membrane proteins is unlike conventional membrane protein methods in that: (i) the method generates sufficient and soluble protein yields for functional and structural studies; (ii) the process is high throughput–amenable; and is (iii) compatible with a variety of cell-free lysates; (iv) native membrane protein may be expressed; and (v) the final complex is freely accessible from both sides of the lipid bilayer.
